PROJECT SUMMARY T cell exhaustion is a common feature of chronic infections and cancer that limits viral or tumor control. A major breakthrough was the demonstration that T cell exhaustion could be reversed by targeting inhibitory receptors such as PD1 and LAG3 and that synergy exists between these receptors. Recent cancer clinical trials demonstrated robust responses following targeted blockade of PD1 with additional trials underway for LAG3. Despite the clinical promise, these inhibitory receptors also control tolerance and autoimmunity and combined genetic deficiency in PD1 and LAG3 in mice results in severe multiorgan autoimmunity. Little is known about the cellular and molecular mechanism by which PD1 and LAG3, and the synergy between them, regulates T cell exhaustion versus autoimmunity. Moreover, it is unclear whether blockade in vivo has similar effects on CD8+ T cells and CD4+ T cells or whether different aspects of exhaustion are regulated by these receptors in each lineage. However, defining the cellular and molecular mechanisms of by which these receptors operate independently and synergistically is critical to understanding the balance between effective immunity to chronic infections and tumors and self-tolerance. Thus, our hypothesis is that the synergy between the PD1 and LAG3 pathways operates via a combination of cellular and molecular mechanisms that differ between CD8+ and CD4+ T cells during chronic viral infection. We will test this hypothesis in the following Aims: AIM 1: Does disruption of PD1 and LAG3 signals cooperate for molecular as well as cellular synergy in the reversal of CD8+ T cell exhaustion during chronic viral infection? Thus, we will use novel genetic strategies combined with blockade in vivo during chronic viral infection to test: (1) whether individual or combined signals from these pathways regulate the induction of CD8+ T cell exhaustion; (2) whether PD1 and/or LAG3 have extrinsic as well as intrinsic roles in regulating established CD8+ T cell exhaustion and define whether cell intrinsic synergy exist for these inhibitory receptors; (3) if synergy is due to preferential reinvigoration of distinct subsets of exhausted CD8+ T cells; and (4) whether key transcriptional changes defining the combinatorial signals from PD1 and LAG3 can define molecular mechanisms of synergy. AIM 2: Do PD1 and LAG3 signals control the development and reversibility of CD4+ T cell exhaustion during chronic viral infection through molecular and/or cellular pathways? Specifically, we will test: (1) whether these pathways are required for the development of CD4+ T cell exhaustion; (2) whether they control established CD4+ T cell exhaustion and define whether PD1 and LAG3 synergize to control CD4+ T cell exhaustion; (3) if subpopulations of exhasuted CD4+ T cells have different depenency on PD1 and LAG3; and (4) whether specific molecular pathways downstreatm of PD1 and/or LAG3 control CD4+ T cell exhaustion.